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Article Two Expansin Genes, AtEXPA4 and AtEXPB5, Are Redundantly Required for Tube Growth and AtEXPA4 Is Involved in Primary Elongation in thaliana

Weimiao Liu 1,2, Liai Xu 1,2 , Hui Lin 3 and Jiashu Cao 1,2,4,*

1 Laboratory of Cell and Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China; [email protected] (W.L.); [email protected] (L.X.) 2 Key Laboratory of Horticultural Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China 3 Crop Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; [email protected] 4 Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Hangzhou 310058, China * Correspondence: [email protected]; Tel.: +86-131-8501-1958

Abstract: The growth of plant cells is inseparable from relaxation and expansion of cell walls. Expansins are a class of binding proteins, which play important roles in the relaxation of cell walls. Although there are many members in expansin gene family, the functions of most expansin genes in plant growth and development are still poorly understood. In this study, the functions of two expansin genes, AtEXPA4 and AtEXPB5 were characterized in . AtEXPA4 and AtEXPB5 displayed consistent expression patterns in mature pollen grains and pollen tubes, but AtEXPA4 also showed a high expression level in primary . Two single mutants, atexpa4

 and atexpb5, showed normal reproductive development, whereas atexpa4 atexpb5 double mutant  was defective in pollen tube growth. Moreover, AtEXPA4 overexpression enhanced primary root

Citation: Liu, W.; Xu, L.; Lin, H.; Cao, elongation, on the contrary, knocking out AtEXPA4 made the growth of primary root slower. Our J. Two Expansin Genes, AtEXPA4 and results indicated that AtEXPA4 and AtEXPB5 were redundantly involved in pollen tube growth and AtEXPB5, Are Redundantly Required AtEXPA4 was required for primary root elongation. for Pollen Tube Growth and AtEXPA4 Is Involved in Primary Root Keywords: Arabidopsis thaliana; expansin genes; AtEXPA4; AtEXPB5; pollen tube growth; root elongation Elongation in Arabidopsis thaliana. Genes 2021, 12, 249. https://doi.org/ 10.3390/genes12020249 1. Introduction Academic Editor: Christian Chevalier Plant growth and development are inseparable from cell proliferation and expansion. Received: 8 December 2020 Different from animal cells, plant cells are surrounded by cell walls, which are highly Accepted: 5 February 2021 Published: 10 February 2021 dynamic and complex networks [1]. Cells are always in a dynamic balance between the expansion of protoplasts and the restraint of cell walls, undergoing irreversible growth [2].

Publisher’s Note: MDPI stays neutral Although cell walls expand slowly while grow, some organs and tissues require with regard to jurisdictional claims in rapid expansion of cell walls during specific periods, such as fast-growing roots and published maps and institutional affil- pollen tubes. iations. Numerous cell wall synthesis and remodeling genes have been reported to be involved in cell wall expansion [3–9], including expansin genes. Expansins can weaken non-covalent bonds between cell wall polysaccharides, thereby promoting slippage and relaxation of microfibers, resulting in cell wall extension and cell expansion [10,11]. Expansins usually have two typical domains, DPBB_1 and Pollen_allerg_1, and most of expansins Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. have signal peptides [12]. DPBB_1 domain is often related to family-45 glycosyl hydro- This article is an open access article lase (GH45), and contains a conserved region that has the double-psi beta-barrel (DPBB) distributed under the terms and fold [13,14]. Although DPBB_1 is similar to GH45, expansins lack the β-1,4-glucanase ac- conditions of the Creative Commons tivity of GH45 enzymes, which in turn lacks the wall expanding activity of expansins [15]. Attribution (CC BY) license (https:// Pollen_allerg_1 is often a pollen allergen, which is found at the C-terminus of expansins [16]. creativecommons.org/licenses/by/ The expansin gene family with many members can be divided into two major subfamilies, 4.0/). namely EXPA and EXPB subfamilies [15]. The various temporal and spatial expression

Genes 2021, 12, 249. https://doi.org/10.3390/genes12020249 https://www.mdpi.com/journal/genes Genes 2021, 12, 249 2 of 16

patterns of expansin genes imply that they perform corresponding functions in different developmental stages [12,17–19]. Many studies have shown that EXPA subfamily plays complex and diverse functions in plant growth and development. For example, Arabidop- sis thaliana EXPA2 (AtEXPA2) participates in under the regulation of upstream transcription factors [20–22]. AtEXPA5 participates in plant growth and develop- ment regulated by ethylene and [23,24]. AtEXPA5 and Oryza sativa EXPA8 (OsEXPA8)[25,26] are both involved in the development of primary roots. The inhibition of their expressions reduces the length of primary root. Moreover, AtEXPA14 and AtEXPA17 participate in the growth of lateral roots and root hairs by the regulation of -related transcription factor LBD18 [27,28]. However, researches on the roles of expansins in plant reproductive development are relatively limited. It was only confirmed that Zea mays EXPB1 (ZmEXPB1) showed a great influence on the growth of pollen tubes in vivo and positively affected the entry of into [29–31]. A previous study showed that two expansin genes, AtEXPA4 and AtEXPB5, were strongly expressed in dry pollen grains, imbibed pollen grains, and pollen tubes [32–36]. Here, we further confirmed the expression patterns of AtEXPA4 and AtEXPB5. By exploring phenotypes of mutants and overexpression lines, we found that AtEXPA4 and AtEXPB5 are redundantly involved in pollen tube elongation, and AtEXPA4 plays a positive role in primary root growth.

2. Materials and Methods 2.1. Plant Materials and Growth Conditions All transgenic plants used in this study were of Arabidopsis thaliana (A. thaliana) Columbia ecotype (Col-0) background and were obtained by Agrobacterium tumefaciens- mediated floral dip method [37]. The homozygous mutants atexpa4 and atexpb5 were obtained by CRISPR/Cas9 system [38]. Two off-target sites of AtEXPA4 and AtEXPB5 were predicted by the CRISPR-P 2.0 website (http://crispr.hzau.edu.cn/CRISPR2/ (accessed on 1 February 2020)), respectively. To obtain heterozygous double mutants of AtEXPA4 and AtEXPB5, atexpb5 and atexpa4 homozygous plants were used as female and male parents in a cross. The homozygous double mutants, atexpa4expb5, were generated by self-crossing with heterozygous F1 plants. The genotypes of 362 F2 plants were confirmed by PCR and sequencing, and genotype statistics and analysis were performed. A 1522-bp promoter sequence and the coding sequence of AtEXPA4 (splicing vari- ant: At2g39700.1) were amplified from Col-0 genomic DNA and inflorescence cDNA. A 1571-bp promoter sequence and the coding sequence of AtEXPB5 (splicing variant: At3g60570.1) were also amplified from Col-0 genomic DNA and inflorescence cDNA. Then they were subcloned into pBI101 vectors to create the fusion overexpression con- structs proAtEXPA4::EXPA4, proAtEXPB5::EXPB5 and the promoter analysis construct proAtEXPA4::GUS, proAtEXPB5::GUS. The complemented lines were obtained by trans- forming proAtEXPA4::EXPA4 and proAtEXPB5::EXPB5 into the double mutant atexpa4expb5, and named atexpa4expb5A4OE and atexpa4expb5B5OE, respectively. The homozygous overex- pression lines AtEXPA4OE, AtEXPB5OE and promoter analysis lines of AtEXPA4, AtEXPB5 −1 were identified from screening T3 plants on plates containing kanamycin (50 mg·L ). Two complementary lines were identified from screening T1 plants on plates containing kanamycin (50 mg·L−1). All seedlings were transplanted into soil, and grown in a 22 ◦C climate chamber under long-day conditions (16 h light/8 h dark). All the primers were listed in Table S1. For root phenotype analysis, the of wild-type, atexpa4, atexpb5, AtEXPA4OE, and AtEXPB5OE were surface-sterilized with 75% ethanol for 5 min and washed 5 times with sterile water. Subsequently, these seeds were geminated on 1/2 Murashige and Skoog solid media and placed horizontally at 4 ◦C for 3 days. Next, plates were transferred to an artificial climate chamber and placed vertically on the platform, and then germinated at 22 ± 1 ◦C under 12 h light/12 h dark cycle. Genes 2021, 12, 249 3 of 16

2.2. mRNA Expression Analysis Trizol reagent (Invitrogen, Carlsbad, CA, USA) were used to extract total RNA from plant tissues and first-strand cDNA was synthesized by PrimerScript RT reagent Kit (TaKaRa, Kyoto, Japan) from 1 µg total RNA. The relative expression levels of corre- sponding genes in different tissues and organs were detected by quantitative real-time PCR (qRT-PCR), which were performed by using TaKaRa TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) on a Real-Time PCR machine (CFX96 Real-Time System, Bio-Rad, Carlsbad, CA, USA). Primers for AtEXPA4 and AtEXPB5 were listed in Supplemental Table S1. BETA-TUBULIN4 (At5g44340) was used as the reference gene for AtEXPA4 and AtEXPB5 expression in different tissues and different transgenic lines. All experiments were performed three biological replicates, and each biological replicate was performed three technical replicates. Moreover, transcript levels of target genes were calculated relative to BETA-TUBULIN4 using the 2−∆∆Ct method.

2.3. Histochemical GUS Staining Assay to Analyze Promoter Activity The promoter analysis constructs, proAtEXPA4::GUS and proAtEXPB5::GUS, were transferred into wild-type plants by Agrobacterium tumefaciens-induced floral dip method. More than six independent T1 lines were screened continuously until T3 homozygous plants were obtained. Seedlings and inflorescences from homozygous plants were used to detect GUS activity. The inflorescences from 35-day-old plants and seedlings at different stages were stained with GUS working solution (GUS working solution: X-Gluc solution and Basic solution (1:9, v/v). X-Gluc solution: dissolving X-Gluc with N-N-dimethylamide (DMF) to make a 20 mM solution. Basic solution: 50 mM NaH2PO4, 50 mM Na2HPO4, 10 mM Na2EDTA, 0.1% (v/v) Triton X-100, 0.5 mM K3 [Fe(CN)6]), and 0.5 mM K4 [Fe(CN)6].) and then incubating in dark at 37 ◦C overnight. Tissues were decolorized in 75% and 90% ethanol in order, and images were taken by a differential interference microscopy (Nikon, Tokyo, Japan). Each staining contained at least three biological replicates. Moreover, the stage of flower development was confirmed by previous studies [39,40].

2.4. Subcellular Localizations of AtEXPA4 and AtEXPB5 To observe subcellular distributions of AtEXPA4 and AtEXPB5, we amplified the AtEXPA4 and AtEXPB5 coding sequences with gene-specific primers (Table S1), and then subcloned these into a vector containing the enhanced green fluorescent genes (eGFP), to form pro35S::AtEXPA4::eGFP and pro35S::AtEXPB5::eGFP constructs. The fusion vectors were transiently transformed into onion epidermal cells using a helium-driven accelera- tor (PDS/1000, Bio-Rad). The parameters were as follows: 1 µm gold particles, 1100 psi bombardment pressure, and a distance of 9 cm from microcarrier to samples. After 24 h of cultivation, the onion epidermal cells with eGFP expression were observed and pictures were taken by a fluorescence microscope (ECLIPSE 90i, Nikon). To visualize eGFP distri- bution, onion epidermal cells were plasmolyzed in 0.1 g·mL−1 sucrose solution for 5 min. For subcellular localization experiments using tobacco (Nicotiana benthamiana), the fusion vectors were transiently transformed into epidermal cells by the infiltrated method. After 48 h of introduction, the subcellular localization of eGFP-fusion protein was analyzed by a confocal laser scanning microscope (A1, Nikon) with the NIS-elements AR software version 4.60(Nikon). The subcellular localization experiments of AtEXPA4 and AtEXPB5 have been carried out using at least three biological replicates, and no less than 5 cells with eGFP signal were observed in each replicate.

2.5. Analysis of Primary Root and Length To observe primary root growth, the 3, 5, 7-day-old seedlings were taken photos by a stereo microscope (Leica, Germany). Subsequently, the primary root length was measured ranging from the base of hypocotyl to the root tip by ImageJ 1.52a software (https://imagej.nih.gov/ij/ (accessed on 29 March 2020)). Genes 2021, 12, 249 4 of 16

The length of root meristem was defined as the distance from quiescent center (QC) to transition zone (TZ, that is the position of the first elongating cortical cell) [4]. To measure the length of root , roots were fixed in saturated chloral hydrate and images were taken by a differential interference microscopy (Nikon, Japan). All experiments were performed in three biological replicates with at least 12 seedlings measured in each replicate.

2.6. Phenotype Analysis of Pollen and Pollen Tube Alexander staining was used to detect pollen vitality [41]. Mature pollen grains that develop normally will be dyed red or purple, and abnormal or immature pollen grains will be dyed blue or green. 40,6-Diamidino-2-phenylindole (DAPI) staining was used to detect whether pollen nuclei were normal [42]. Under ultraviolet light, normal pollen nuclei (two nuclei and one nutrient nucleus) will produce intense fluorescence. Aniline blue staining could detect callose development and degradation [43]. In tetrad stage, are wrapped by callose wall, which can combine with the aniline blue dye to produce strong fluorescence under ultraviolet illumination. Under normal circumstances, the callose wall gradually degrades with the development of pollen. In mature pollen stage, the callose wall has been completely degraded and only the weak fluorescence of pollen grains can be observed but no strong fluorescence will appear under ultraviolet light. All micrographs were taken by an inverted fluorescence microscope (Nikon, Japan). Scanning electron microscope (SEM) observation and pollen germination in vivo were conducted as described in Lin et al. [44]. The mature pollens and tetrads were peeled off from flower in stage 13 and stage 6–8 of 35-d-old inflorescences, respectively. Each kind of staining was repeated three times, using at least 5 plants each time, and observing no less than 30 fields of view. The length of pistil and pollen tube was measured by ImageJ, and the ratio of pollen tube to pistil length was calculated as an indicator of the germination degree of pollen tube. The experiments were carried out using three biological replications with at least 7 pistils measured in each replicate.

3. Results 3.1. Analysis of AtEXPA4 and AtEXPB5 Expression Patterns Through the analysis of domains on the Pfam website (http://www.sanger.ac.uk/ Software/Pfam/ (accessed on 21 March 2018)), we confirmed that both AtEXPA4 and AtEXPB5 contain DPBB_1 and Pollen_allerg_1, which have typical domains of the expansin gene family (Figure S1) [12].qRT-PCR and GUS reporting system were used to explore spatial and temporal expression of them. The results showed that AtEXPA4 seemed to expressed ubiquitously (Figure1A), but its expression level was higher in inflorescences and roots (Figure1A,B). In inflorescences, AtEXPA4 was expressed from the stage 11 of floral buds to the mature pollen stage. Subsequently, strong GUS signal appeared in pollen tubes, which indicated that AtEXPA4 was also highly expressed during pollen tube growth (Figure1C). Further research found that AtEXPA4 was expressed in the root tips, especially in root meristems of 3-d-old seedlings. As time passed, AtEXPA4 was expressed in the entire root by the 7th day after seed germination (Figure1D). As for AtEXPB5, the qRT-PCR result showed that AtEXPB5 was predominantly expressed in inflorescences and siliques, but weakly expressed in other tissues (Figure1E,F). GUS staining also confirmed that AtEXPB5 was highly expressed in inflorescences. Moreover, GUS signal in pollen tube was very significant, suggesting that AtEXPB5 was strongly expressed during pollen tube growth (Figure1G). Genes 2021, 12, x FOR PEER REVIEW 5 of 16

AtEXPB5 was predominantly expressed in and siliques, but weakly ex- pressed in other tissues (Figure 1E,F). GUS staining also confirmed that AtEXPB5 was highly expressed in inflorescences. Moreover, GUS signal in pollen tube was very sig- nificant, suggesting that AtEXPB5 was strongly expressed during pollen tube growth Genes 2021, 12, 249 5 of 16 (Figure 1G).

FigureFigure 1. Expression pattern pattern analysis analysis of of AtEXPA4AtEXPA4 and AtEXPB5..( (AA,,EE)) qRT-PCR qRT-PCR analysis analysis of of AtEXPA4AtEXPA4 ((A)) and AtEXPB5 ((E)) transcripts transcripts in in different different tissues tissues of of Arabidopsis thaliana thaliana:: 35-d-old 35-d-old roots roots (R), (R), 35-d-old 35-d-old stems stems (Ste), (Ste), young young rosette leaves (L), (L), 35-d-old35-d-old inflorescences inflorescences (Inf)(Inf) andand siliquessiliques (Si). (Si).BETA-TUBULIN4 BETA-TUBULIN4was was used used as as the the reference reference gene. gene.AtEXPA4 AtEXPA4expression expression in leaf in leafand andAtEXPB5 AtEXPB5expression expression in stem in stem were were normalized normalized to 1. Theto 1. values The valu arees the are mean the ±meanSD (standard± SD (standard deviation), deviation), three biologicalthree bi- ological replicates with three technical replicates in each biological replicate. (B–D) Analysis of proAtEXPA4::GUS activ- replicates with three technical replicates in each biological replicate. (B–D) Analysis of proAtEXPA4::GUS activity. (B) GUS ity. (B) GUS activity in inflorescences. (C) GUS activity in different developmental stages of buds and pollen tubes. (D) activity in inflorescences. (C) GUS activity in different developmental stages of buds and pollen tubes. (D) GUS activity in GUS activity in seedlings at different time points after germination, days (d). (F,G) Analysis of proAtEXPB5::GUS activity. (seedlingsF) GUS activity at different in inflorescences. time points after (G) germination,GUS activity daysin different (d). (F ,developmentalG) Analysis of proAtEXPB5 stages of buds,::GUS stigmas,activity. pollen (F) GUS tubes, activity and 7-d-oldin inflorescences. seedlings. (ScaleG) GUS bars activity of pollen in differenttubes, 25 developmental μm. Scale bars stagesof others, of buds, 1 mm. stigmas, pollen tubes, and 7-d-old seedlings. Scale bars of pollen tubes, 25 µm. Scale bars of others, 1 mm. 3.2. AtEXPA4 and AtEXPB5 Exhibit Cell Wall Localizations 3.2. AtEXPA4To test whether and AtEXPB5 AtEXPA4 Exhibit and Cell AtEXPB Wall Localizations5 were cell wall binding proteins, eGFP genesTo were test fused whether to AtEXPA4 and AtEXPB5AtEXPB5 werecoding cell sequences wall binding to form proteins, AtEXPA4eGFP::geneseGFP andwere AtEXPB5 fused to::eGFPAtEXPA4 constructsand AtEXPB5 under thecoding control sequences of constitutive to form CaMVAtEXPA4 35S ::promoter.eGFP and Subsequently,AtEXPB5::eGFP theseconstructs constructs under were the transien controltly of constitutivetransformedCaMV into onion 35S promoter. and tobacco Subse- ep- idermalquently, cells. these The constructs eGFP fluorescence were transiently in toba transformedcco leaf epidermal into onion cells and appeared tobacco epidermal in nuclei andcells. plasma The eGFP membrane, fluorescence which in indicated tobacco leaf that epidermal AtEXPA4 cells and appeared AtEXPB5 in localized nuclei and in plasmanuclei andmembrane, plasma membrane which indicated (Figure that S2). AtEXPA4 Furthermor ande, AtEXPB5 the eGFP localized fluorescent in nuclei signal and associated plasma withmembrane AtEXPA4 (Figure and S2). AtEXPB5 Furthermore, localized the eGFPhomogeneously fluorescent signalin unplasmolyed associated with transformed AtEXPA4 onionand AtEXPB5 cells, including localized homogeneously and nuclei in unplasmolyed (Figure 2E,F,I,J). transformed In plasmolyed onion cells, cells, includ- the eGFPing cytoplasm fluorescence and not nuclei only (Figure appeared2E,F,I,J). in cyto In plasmolyedplasm, nuclei, cells, and the cell eGFP membrane, fluorescence but also not distributedonly appeared on the in cytoplasm,cell wall (Figure nuclei, 2G,H,K,L). and cell membrane,On the contrary, but also there distributed was no eGFP on the signal cell onwall the (Figure cell wall2G,H,K,L). in the control On the contrary,cells after there plasmolysis was no eGFP(Figure signal 2C,D). on theThese cell results wall in con- the firmedcontrol that cells AtEXPA4 after plasmolysis and AtEXPB5 (Figure exhibited2C,D). These cell wall results localizations. confirmed that AtEXPA4 and AtEXPB5 exhibited cell wall localizations. 3.3. Identification of atexpa4, atexpb5, atexpa4expb5 Mutants, and AtEXPA4OE, AtEXPB5OE 3.3. Identification of atexpa4, atexpb5, atexpa4expb5 Mutants, and AtEXPA4OE, Lines AtEXPB5OE Lines In order to characterize the functions of AtEXPA4 and AtEXPB5 in plant growth In order to characterize the functions of AtEXPA4 and AtEXPB5 in plant growth and and development, gene knockout and overexpression methods were used. The promoter development, gene knockout and overexpression methods were used. The promoter of Cas9 of Cas9 was replaced with YAOZHE promoter (proAtYAO) for improving the editing ef- was replaced with YAOZHE promoter (proAtYAO) for improving the editing efficiency of CRISPR/Cas9 system (Figure S3A) [45]. Two sgRNAs targeting AtEXPA4 or AtEXPB5 were designed respectively to obtain knockout lines. For AtEXPB5, both sgRNAs worked, while AtEXPA4 only sgRNA11 of worked (Figures3A and S4). Subsequently, homozygous lines atexpa4-6/47/149 and atexpb5-4/8/9 were used for phenotypic observation (Figure3B,C) . We also checked whether these lines were off-target by PCR and sequencing methods. The results showed that none of the 33 atexpa4 and 47 atexpb5 plants at off-target sites Genes 2021, 12, x FOR PEER REVIEW 6 of 16 Genes 2021, 12, x FOR PEER REVIEW 6 of 16

ficiency of CRISPR/Cas9 system (Figure S3A) [45]. Two sgRNAs targeting AtEXPA4 or ficiency of CRISPR/Cas9 system (Figure S3A) [45]. Two sgRNAs targeting AtEXPA4 or AtEXPB5 were designed respectively to obtain knockout lines. For AtEXPB5, both sgR- AtEXPB5 were designed respectively to obtain knockout lines. For AtEXPB5, both sgR- NAs worked, while only sgRNA11 of AtEXPA4 worked (Figures 3A and S4). Subse- NAs worked, while only sgRNA11 of AtEXPA4 worked (Figures 3A and S4). Subse- quently, homozygous lines atexpa4-6/47/149 and atexpb5-4/8/9 were used for phenotypic Genes 2021, 12, 249 quently, homozygous lines atexpa4-6/47/149 and atexpb5-4/8/9 were used for phenotypic6 of 16 observation (Figure 3B,C). We also checked whether these lines were off-target by PCR observation (Figure 3B,C). We also checked whether these lines were off-target by PCR and sequencing methods. The results showed that none of the 33 atexpa4 and 47 atexpb5 and sequencing methods. The results showed that none of the 33 atexpa4 and 47 atexpb5 plants at off-target sites were edited (Tables S2 and S3). Moreover, homozygous double plantswere edited at off-target (Tables sites S2 and were S3). edited Moreover, (Tables homozygous S2 and S3). doubleMoreover, mutant homozygousatexpa4expb5 doublewas mutant atexpa4expb5 was screened by PCR and sequencing (Figure S5). In addition, mutantscreened atexpa4expb5 by PCR and was sequencing screened (Figure by PCR S5). and In addition, sequencingAtEXPA4 (Figureand S5).AtEXPB5 In addition,were AtEXPA4 and AtEXPB5 were overexpressed under the control of their own promotersOE AtEXPA4overexpressed and AtEXPB5 under the were control overexpressed of their own under promoters the control (Figure of S3B,C). their ForownAtEXPA4 promoters, (Figure S3B,C). For AtEXPA4OE, seven overexpression lines were generated and they all (Figureseven overexpression S3B,C). For AtEXPA4 lines wereOE, seven generated overexpression and they all lines showed were 5.8–37.1-fold generated and increase they all in showed 5.8–37.1-fold increase in roots and inflorescences compared with wild-type. showedroots and 5.8–37.1-fold inflorescences increase compared in roots with and wild-type. inflorescences compared with wild-type.

FigureFigure 2. SubcellularSubcellular localizations localizations of of AtEXPA4::eGFP AtEXPA4::eGFP and and AtEXPB5::eGFP AtEXPB5::eGFP fusion fusion proteins proteins in in onion onion epidermal epidermal cells. cells. Figure 2. Subcellular localizations of AtEXPA4::eGFP and AtEXPB5::eGFP fusion proteins in onion epidermal cells. ((AA––DD),), Control Control cells with eGFP signals. ( (E––HH),), Onion Onion epidermal cells cells with with AtEXPA4::eGFP fusion signals. ( (II––LL),), Onion Onion (A–D), Control cells with eGFP signals. (E–H), Onion epidermal cells with AtEXPA4::eGFP fusion signals. (I–L), Onion epidermalepidermal cells with AtEXPB5::eGFP fusionfusion signals.signals. ((AA,,CC,,EE,,GG,,II,,KK),), FluorescenceFluorescence images.images. ((BB,D,D,F,F,H,H,J,J,L,L),), Brightfield Brightfield images. imag- epidermal cells with AtEXPB5::eGFP fusion signals. (A,C,E,G,I,K), Fluorescence images. (B,D,F,H,J,L), Brightfield imag- es.(A ,(BA,E,B,F,E,I,,FJ,),I,J Unplasmolysed), Unplasmolysed cells. cells. (C (,CD,,DG,G,H,H,K,,KL,),L), Plasmolysed Plasmolysed cells. cells. Arrows Arrows andand arrowarrow headsheads indicateindicate cell walls and and es. (A,B,E,F,I,J), Unplasmolysed cells. (C,D,G,H,K,L), Plasmolysed cells. Arrows and arrow heads indicate cell walls and cytoplasm, respectively. Scale bars, 100 μm. cytoplasm, respectively. ScaleScale bars,bars, 100100 µμm.m.

Figure 3. Confirmation of AtEXPA4 and AtEXPB5 transgenic plants. (A), Genomic location of sgRNAs targeting to Figure 3.3. ConfirmationConfirmation of ofAtEXPA4 AtEXPA4and andAtEXPB5 AtEXPB5transgenic transgenic plants. plants. (A), Genomic (A), Genomic location location of sgRNAs of sgRNAs targeting targeting to AtEXPA4 to AtEXPA4 and AtEXPB5. PAM sites are highlighted in red. (B,C), the gene editing situation of AtEXPA4 and AtEXPB5 by AtEXPA4and AtEXPB5 and. AtEXPB5 PAM sites. arePAM highlighted sites are highlighted in red. (B,C ),in the red. gene (B,C editing), the gene situation editing of AtEXPA4 situationand of AtEXPA4AtEXPB5 andby CRISPR/Cas9 AtEXPB5 by CRISPR/Cas9 system in T3 plants. Underlined letters, red letters, and blue letters represent PAM sites, gene editing sites, CRISPR/Cas9 system in T3 plants. Underlined letters, red letters, and blue letters represent PAM sites, gene editing sites, andsystem stop in codons, T3 plants. respectively. Underlined (D letters,), qRT-PCR red letters, analysis and of blue AtEXPA4 lettersrepresent expression PAM in AtEXPA4 sites, geneOE editing lines. 35-d-old sites, and roots stop (R) codons, and and stop codons, respectively. (D), qRT-PCR analysis of AtEXPA4 expressionOE in AtEXPA4OE lines. 35-d-old roots (R) and inflorescencesrespectively. ( D(Inf).), qRT-PCR BETA-TUBULIN4 analysis of AtEXPA4was usedexpression as the reference in AtEXPA4 gene. AtEXPA4lines. 35-d-old expression roots (R)in control and inflorescences lines were nor- (Inf). inflorescences (Inf). BETA-TUBULIN4 was used as the reference gene. AtEXPA4 expression in control lines were nor- malizedBETA-TUBULIN4 to 1. (E),was qRT-PCR used as analysis the reference of AtEXPB5 gene. AtEXPA4 expressionexpression in AtEXPB5 in controlOE lines. lines were35-d-old normalized inflorescences to 1. (E ),(Inf). qRT-PCR BE- malized to 1. (E), qRT-PCR analysis of AtEXPB5 expression in AtEXPB5OE lines. 35-d-old inflorescences (Inf). BE- analysis of AtEXPB5 expression in AtEXPB5OE lines. 35-d-old inflorescences (Inf). BETA-TUBULIN4 was used as the reference gene. AtEXPB5 expression in control lines were normalized to 1. The values are the mean ± SD, three biological replicates with three technical replicates in each biological replicate.

Genes 2021, 12, 249 7 of 16

As for AtEXPB5OE, a total of 7 overexpression lines were obtained, which showed an increase of 15.0–38.5-fold compared with the wild type. The growth of homozygous lines with the highest overexpression, AtEXPA4OE-4 and AtEXPB5OE-1 were subsequently monitored (Figure3D,E).

3.4. Varied Expressions of AtEXPA4 and AtEXPB5 Show No Effect on and Pollen Vitality The results of morphological observation showed that whether AtEXPA4 and AtEXPB5 were overexpressed or knocked out, plant overall morphology was not affected. Moreover, , , and pistils were not significantly abnormal compared with wild- type. Also, double mutant, atexpa4expb5, was not different from control lines (Figure S6). Furthermore, pollen development of transgenic plants was observed by cytological staining. Alexander staining revealed that pollen grains produced by these transgenic plants all showed high vitality. By aniline blue staining, they all showed normal thickening of callose during the tetrad stage, and no abnormality in callose degradation during the subsequent pollen maturity stage. DAPI staining was used to observe pollen nuclei, and the result displayed that all transgenic pollen nuclei were development normally. SEM observation revealed that pollen morphology and surface decoration of all transgenic lines were no different from those of wild-type. (Figure S7). These results indicated that the single and double mutations and overexpression of AtEXPA4 and/or AtEXPB5 had no effect on pollen development.

3.5. AtEXPA4 and AtEXPB5 Are Redundantly Required for Pollen Tube Growth To investigate the impacts of AtEXPA4 and AtEXPB5 on pollen germination, in vivo germination assay was performed. After 3 h of pollen germination, wild-type pollen tube length accounted for 34.59% of total pistil length, while the pollen tube length of at- expa4expb5 only accounted for 17.04% of pistil length. The pollen tube length of atexpa4expb5 was 50.74% less than that of wild-type (Figure4A,B). With pollen tube development, the dif- ferences in pollen tube length between double mutants and wild-type became smaller than that in the 3 h after pollen germination, but they were still at significant levels. (Figure4B). By 24 h after , wild-type pollen tubes have been extended to the bottom of pistils, while pollen tube length of double mutants have only grown to 76.45% of pistil (Figure4A,B). These results indicated that the pollen tube elongation of atexpa4expb5 was significantly slower than that of wild-type after self-pollination. However, no difference was observed in atexpa4 and atexpb5 when compared with wild-type at various time points after pollination (Figure4). However, the overexpression of AtEXPA4 and AtEXPB5 had no significant effect on pollen germination and elongation (Figure S8). In addition, we investigated whether AtEXPA4 or AtEXPB5 could complement the double mutant phenotype by observing the pollen germination in vivo of complementary lines. The results of qRT-PCR showed that expression levels of AtEXPA4 and AtEXPB5 in corresponding complementary lines were increased (Figure5A,B). We selected the highest expression lines atexpa4expb5A4OE-18/20/22 and atexpa4expb5B5OE-4/15/17 for pollen germination in vivo. The results indicated that whether transformed the double mutants with AtEXPA4 or AtEXPB5 could complement the slow pollen tube growth (Figure5C,D). Nevertheless, the silique and seed development of either two single mutants or double mutant were not different from that of wild-type (Figure S6), which implied that knocking out AtEXPA4 and AtEXPB5 simultaneously did not affect the transmission of sperm cells and process. We also calculated and analyzed the segregation ratio of F2 plants produced by self-crossing of F1 plants [expa4(+/−) expb5(+/−)], and the results showed that the slower growth did not cause expansin deficient pollen to be less competitive than wild-type pollen (Table1). In summary, AtEXPA4 and AtEXPB5 were redundantly involved in pollen tube germination and elongation, but showed no effect on silique and seed development. Genes 2021, 12, x FOR PEER REVIEW 8 of 16

gation ratio of F2 plants produced by self-crossing of F1 plants [expa4(+/−) expb5(+/−)], and the results showed that the slower growth did not cause expansin deficient pollen to be less competitive than wild-type pollen (Table 1). In summary, AtEXPA4 and AtEXPB5 Genes 2021, 12, 249 were redundantly involved in pollen tube germination and elongation, but showed8 of 16 no effect on silique and seed development.

FigureFigure 4. AtEXPA4 4. AtEXPA4and AtEXPB5and AtEXPB5are involved are involved in pollen in pollen tube growth.tube growth. (A), Aniline (A), Aniline blue staining blue staining of pollen of pollen tubes attubes 3 h, at 12 3 h, h, 12 andh, 24 and h after 24 h pollination after pollination in single in and single double and mutants,double mutants, hours (h). hours Asterisks (h). Asterisks show positions show positions where pollen where tubes pollen arrive, tubes scale arrive, bars,scale 400 µbars,m. ( B400), the μm. percentage (B), the percentage of pollen tube of pollen to pistil tube length to pistil in (A ).length Values in are (A). means, Values error are barsmeans, are error SD, n bars= 12 are pistils SD, per n = 12 replicate,pistils three per replicate, biological thr replicates,ee biologicalt-tests replicates, as ** p < t 0.01.-tests as ** P < 0.01.

Table 1. Segregation data from self-crossing of heterozygous F1 double mutants [expa4(+/−) expb5(+/−)]. Table 1. Segregation data from self-crossing of heterozygous F1 double mutants [expa4(+/−) expb5(+/−)].

2 a 2 χ Test fora 9:3:3:1 TheThe Observed Observed NumberThe The Expected Expected Number of F2 Degrees of Free-χ Test for 9:3:3:1 Genotype of F2 Plants2 Degrees of Freedom 2 Genotype of F PlantsNumber of F Plants Number of F Plants 2 2 Expected χ (p of2 F2 Plants 2 Plants dom χ Expectedχ2 χ (p < 0.05) < 0.05) expa4(+/)expa4expb5(+/) (+/)expb5(+/) 196196 203.625 203.625 − − expa4( / ) expb5(+/) 76 67.875 b expa4(−/−−) expb5− (+/) 76 67.875 3 4.576 7.815 expa4(+/) expb5( / ) 75 67.875 3 4.576 b 7.815 expa4(−expa4/−) expb5(+/) expb5(−/−()−/−) 1575 22.625 67.875 a 2 χexpa4tests(− were/−) expb5 conducted(−/−) to test a null hypothesis15 that F2 plants segregate 22.625 from each other in a predicted 9:3:3:1 Mendelian ratio. b 2 2 χ a< χ Expected2 tests wereχ indicated conducted that to the test null a null hypothesis hypothesis was accepted,that F2 plants that is, segregate the separation from ratioeach ofothe F2 rplants in a predicted accorded with9:3:3:1 the Mendelian 9:3:3:1 Mendelianb ratio.2 2 ratio. χ < Expected χ indicated that the null hypothesis was accepted, that is, the separation ratio of F2 plants accorded with the 9:3:3:1 Mendelian ratio.

Genes 2021, 12, x FOR PEER REVIEW 9 of 16 Genes 2021, 12, 249 9 of 16

Figure 5.5. AtEXPA4 oror AtEXPB5AtEXPB5 cancan complementcomplement the slow pollen tube growth. ( (AA,,BB),), qRT-PCR qRT-PCR analysis of of AtEXPA4 (A) or AtEXPB5AtEXPB5 (B(B)) expression expression in in 35-d-old 35-d-ol inflorescencesd inflorescences from fromatexpa4expb5 atexpa4expb5, atexpa4expb5, atexpa4expb5A4OEA4OE, and, andatexpa4expb5 atexpa4expb5B5OEB5OE. BETA-. BE- TUBULIN4TA-TUBULIN4was was used used as theas the reference reference gene, gene, and and expression expression in inatexpa4expb5 atexpa4expb5was was normalized normalized to to 1. 1. TheThe valuesvalues areare thethe mean ± SD,SD, three three biological biological replicat replicateses with with three three technical replicates in each biological replicate. (C) Aniline blue staining of pollen tubes at 3 h, 12 h, and 24 h after pollination, hours (h). Asterisks show positions where pollen tubes arrive, scale of pollen tubes at 3 h, 12 h, and 24 h after pollination, hours (h). Asterisks show positions where pollen tubes arrive, scale bars, 300 μm. (D) the percentage of pollen tube to pistil length in (C). Values are means, error bars are SD, n = 7 pistils per bars, 300 µm. (D) the percentage of pollen tube to pistil length in (C). Values are means, error bars are SD, n = 7 pistils per replicate, three biological replicates. replicate, three biological replicates. 3.6. AtEXPA4 Participates in Primary Root Elongation 3.6. AtEXPA4 Participates in Primary Root Elongation The increasing expression of AtEXPA4 in primary roots during seedling develop- mentThe suggested increasing that expression it might be of AtEXPA4involved inin primaryroot growth. roots duringTherefore, seedling we measured development the primarysuggested root that length it might of seedlings. be involved Interestingly, in root growth. when Therefore, compared we with measured the wild-type the primary at 3d, 5d,root and length 7d of after seedlings. seed germination, Interestingly, whenthe length compared of atexpa4 with the primary wild-type root at 3d,decreased 5d, and 7dby after seed germination, the length of atexpa4 primary root decreased by 30.76%,OE 21.92%, and 30.76%, 21.92%, and 37.24%, respectively. On theOE contrary, AtEXPA4 seedlings exhib- ited37.24%, increased respectively. primary On root the length contrary, byAtEXPA4 16.52% andseedlings 9.98% at exhibited3d and 5d increased after seed primary germi- root length by 16.52% and 9.98% at 3d and 5d after seed germination. Although the primary nation. Although the primary root length of AtEXPA4OE only increased by 5.67% com- root length of AtEXPA4OE only increased by 5.67% compared with the wild-type at the pared with the wild-type at the 7th day after germination, it still reached a significant 7th day after germination, it still reached a significant level. (Figure6A,B). Moreover, the level. (Figure 6A,B). Moreover, the mutation and overexpression of AtEXPB5 did not af- mutation and overexpression of AtEXPB5 did not affect the growth and development of fect the growth and development of roots (Figure S9A,B). Notably, the change pattern of roots (Figure S9A,B). Notably, the change pattern of root meristem size was similar to that root meristem size was similar to that of root length. The meristem length of atexpa4 was of root length. The meristem length of atexpa4 was reduced by 25.41%, 18.26%, and 33.00% reduced by 25.41%, 18.26%, and 33.00% compared with the wild-type after 3, 5, and 7 compared with the wild-type after 3, 5, and 7 days of seed germination (Figure7A,B). By days of seed germination (Figure 7A,B). By contrast, the overexpression of AtEXPA4 led contrast, the overexpression of AtEXPA4 led root meristems to increase by 12.15%, 11.27%, root meristems to increase by 12.15%, 11.27%, and 7.51% at 3d, 5d, and 7d after seed and 7.51% at 3d, 5d, and 7d after seed germination (Figure7A,C). germination (Figure 7A,C). In addition, the primary root growth rates of wild-type, atexpa4, and AtEXPA4OE were In addition, the primary root growth rates of wild-type, atexpa4, and AtEXPA4OE similar from the 3rd day to the 5th day after germination (Figure6C). However, some werechanges similar occurred from fromthe 3rd the day 5th dayto the to 5th the da 7thy dayafter after germination germination. (Figure On 6C). the oneHowever, hand, primarysome changes root growth occurred rates from of wild-typethe 5th day and toAtEXPA4 the 7th dayOE were after stillgermination. consistent On but the higher one OE hand,than before. primary On root the othergrowth hand, rates the of primarywild-type root and growth AtEXPA4 rate of wereatexpa4 stillwas consistent slower than but higherthat of wild-typethan before. and OnAtEXPA4 the otherOE (Figurehand, the6C). primary The change root patterngrowth ofrate meristem of atexpa4 growth was OE slowerrate was than consistent that of withwild-type that ofand root AtEXPA4 elongation (Figure rate. That 6C).is, The the change meristem pattern growth of meri- rate stemof each growth transgenic rate was line consistent was closely with resembled that of root in the elongation first 5 days rate. after That seed is, the germination, meristem

Genes 2021, 12, x FOR PEER REVIEW 10 of 16

Genes 2021, 12, 249 10 of 16

growth rate of each transgenic line was closely resembled in the first 5 days after seed whilegermination, the growth while rate the of growthatexpa4 ratewas of significantly atexpa4 was reduced significantly from reduced the 5th day from to the the 5th 7th day (Figureto the 7th7A,C) day. These(Figure findings 7A,C). suggested These findings that AtEXPA4 suggestedmight that exert AtEXPA4 the greatest might effect exert from the greatestthe 5th day effect to thefrom 7th the day 5th after day seed to the germination. 7th day after For seedAtEXPB5 germination., the primary For AtEXPB5 root growth, the primaryrates of atexpb5root growthand AtEXPB5 rates of OEatexpb5were notand significantlyAtEXPB5OE differentwere not fromsignificantly that of wild-type different (Figurefrom that S9C). of wild-type Taken together, (FigureAtEXPA4 S9C). Takencould together, promote AtEXPA4 the elongation could of promote primary the root elon- and gationroot meristem of primary during root root and growth root meristem and development, during root but growthAtEXPB5 andhad development, no effect on but the AtEXPB5growth of had primary no effect root. on the growth of primary root.

Figure 6.6. AtEXPA4AtEXPA4 positivelypositively regulatesregulates primaryprimary rootroot elongation.elongation. ((AA)) AtEXPA4AtEXPA4OEOE andand atexpa4atexpa4 seedlingsseedlings germinatedgerminated andand grown for 3, 5, 5, and and 7 7 days, days, scale scale bars, bars, 2 2mm. mm. (B ()B Root) Root lengths lengths in in(A ().A ().C) ( CRoot) Root growth growth rates rates from from the the3rd 3rdday dayto the to 5th the day 5th dayand andfrom from the the5th 5thday day to the to the 7th 7th day day after after seed seed ge germination.rmination. Values Values are are means, means, error error bars bars are are SD, n ≥ 2525 seedlings per replicate, threethree biologicalbiological replicates,replicates, tt-tests-tests asas **** pP< < 0.010.01. and * p < 0.05.

Genes 2021, 12, x FOR PEER REVIEW 11 of 16 Genes 2021, 12, 249 11 of 16

OE FigureFigure 7.7.AtEXPA4 AtEXPA4positively positively regulates regulates root ro meristemot meristem length. length. (A) Differential (A) Differential interference interference images images of AtEXPA4 of AtEXPA4and atexpa4OE and rootatexpa4 meristem root meristem boundary boundary after seed afte germinatedr seed germinated for 3, 5, for and 3, 7 5, days, and scale7 days, bars, scale 100 bars,µm. 100 (B) μ Rootm. (B meristem) Root meristem lengths lengths in (A). (inC) ( RootA). (C meristem) Root meristem growth rates growth from rates the 3rdfrom day the to 3rd the day 5th dayto the and 5th from day the and 5th from day tothe the 5th 7th day day to afterthe 7th seed day germination. after seed Valuesgermination. are means, Values error are bars means, are SD,errorn ≥bars25 are seedlings SD, n ≥ per 25 seedlings replicate, threeper replicate, biological three replicates, biologicalt-tests replicates, as ** p < t-tests 0.01. as ** P < 0.01. 4. Discussion 4. Discussion During pollen tube growth, cell walls provide mechanical strength resisting turgor pressureDuring to protect pollen twotube sperm growth, cells cell [46 walls]. Numerous provide mechanical cell wall synthesis strength and resisting remodeling turgor genespressure have to beenprotect reported two sperm in this cells process. [46]. Numerous In terms ofcell cell wall wall synthesis synthesis, and mutations remodeling of pollen-expressedgenes have beenA. reported thaliana incellulose this process. synthase-like In terms D genesof cellCSLD1 wall synthesis,and CSLD4 mutationscaused sig- of nificantpollen-expressed reduction inA. cellulosethaliana depositioncellulose syntha of pollense-like tube D wall,genes which CSLD1 disrupted and CSLD4 the geneticcaused transmissionsignificant reduction of male gametophytesin cellulose deposition [3]. Moreover, of pollen two putativetube wall,A. thalianawhich disruptedgalacturono- the syltransferasegenetic transmission genes GAUT13 of male andgametoGAUT14phyteswere [3]. essentialMoreover, for two pollen putative tube growthA. thaliana [47]. Changesgalacturonosyltransferase in cell wall assembly genes are GAUT13 relevant and to pollenGAUT14 tube were mechanical essential properties.for pollen tube For example,growth [47]. suppressing Changes thein cell expression wall assembly of four are leucine-rich relevant repeatto pollen extensin tube mechanical genes (LRX8–11 prop-) compromisederties. For example, pollen germinationsuppressing andthe pollenexpressi tubeon growthof four [leucine-rich46,48]. In terms repeat of cellextensin wall remodeling,genes (LRX8–11 pectin) compromised methylesterase pollen (PME) germination demethylates and pectin pollen to maintain tube growth stable growth[46,48]. ofIn pollenterms tubes.of cell Mutations wall remodeling, of PME genes, pectin such methylesterase as VGD1 [9], AtPPME1(PME) demethylates[49], and AtPME48 pectin[ 6to], showedmaintain retarded stable growth pollen tubeof pollen growth tubes.in vivo Mutationsand in vitro of .PME Additionally, genes, such PME as inhibitorsVGD1 [9], (PMEIs)AtPPME1 are [49], thought and AtPME48 to be key [6], regulators showed of retarded cell wall pollen stability tube at growth the tip ofin pollenvivo and tube. in Suppressingvitro. Additionally, the expression PME inhibitors of Brassica (PMEIs) oleracea are PMEI1 thoughtresulted to be in key partial regulators male sterility of cell andwall decreasedstability at seed the tip set of by pollen inhibition tube of. Suppressing pollen tube growththe expression [50]. Furthermore, of Brassica oleracea polygalactur- PMEI1 onase,resulted which in partial leads tomale degradation sterility ofand pectin decreased and decomposition seed set by ofinhibition cell walls, of has pollen also beentube confirmedgrowth [50]. to playFurthermore, an important polygalacturonase, role in the development which leads of pollen to degradation tubes [7,51 of]. Expansins,pectin and asdecomposition a type of cell wallof cell remodeling walls, has proteins, also been can confirmed respond to to the play rapid an expansion important of role cell in walls the anddevelopment affect pollen of pollen tube growth tubes [7,51]. [52]. Our Expansins, results confirmed as a type thatof cell the wall expressions remodeling of AtEXPA4 proteins, andcan AtEXPB5respond towere the significantlyrapid expansion high of in cell mature walls pollen and affect grains pollen and pollen tube tubes.growth Moreover, [52]. Our proteinsresults confirmed coding by that these the two expressions genes were of located AtEXPA4 on the and cell AtEXPB5 wall and were in the significantly cytoplasm, high and thisin mature was consistent pollen grains with the and positioning pollen tubes. result Mo ofreover, a soybean proteins expansin coding protein by these GmEXPB2 two genes [53]. Twowere single located mutants, on the cellatexpa4 walland andatexpb5 in the cyto, didplasm, not exhibit and this any was observable consistent defects with the in pol- po- lenssitioning and pollenresult tubes,of a soybean but atexpa4expb5 expansinmutant protein was GmEXPB2 defective [53]. in pollen Two tubesingle elongation. mutants, Moreover,atexpa4 and the atexpb5 results, did ofcomplementary not exhibit any experimentsobservable defe showedcts in that pollens whether and transformedpollen tubes, the double mutants with AtEXPA4 or AtEXPB5 could complement the slow pollen tube but atexpa4expb5 mutant was defective in pollen tube elongation. Moreover, the results of

Genes 2021, 12, 249 12 of 16

growth. However, the overexpression of AtEXPA4 and AtEXPB5 did not affect pollen tube growth. We speculate that this may be because the highest multiple of AtEXPA4OE and AtEXPB5OE overexpression line does not exceed 40 folds, which may not be enough to accelerate the rate of pollen tube elongation. Additionally, although the pollen germination rate of the double mutants decreased, the mutation of AtEXPA4 and AtEXPB5 did not affect the pollen competitiveness or the normal development of siliques. Taken together, AtEXPA4 and AtEXPB5 only showed redundant functions in pollen tube growth. Interestingly, there are abundant MYB binding sites on promoters of AtEXPA4 and AtEXPB5 (Figure S10), which indicates that MYB transcription factors may participate in pollen tube and root growth by regulating expressions of AtEXPA4 and AtEXPB5.A previous study showed that a R2R3 MYB factor, TDF1, affected development by directly binding to AtEXPB5 promoter and co-regulated AtEXPB5 with another tran- scription factor AMS [54]. Thence, AtEXPB5 might play a role in tapetum and pollen exine development. However, there was no defect in exine of atexpb5 pollens, suggesting that TDF1 regulated the development of tapetum by acting on other cell wall remodeling genes. Pollen tube germination usually starts from the intine of mature pollen grains [6]. AtEXPA4 and AtEXPB5 were strongly expressed in mature pollen grains and pollen tubes, and simultaneous mutation of them led to obstruction of pollen tube elongation, implying that they might affect pollen tube growth by regulating the development of pollen intine. Moreover, AtMYB4, AtMYB32, AtMYB97, AtMYB101, and AtMYB120 can participate in pollen and pollen tube development [55,56], suggesting that they may regulate expressions of AtEXPA4 and AtEXPB5, but further evidence is needed. Some cell wall-related genes that affect pollen tube growth also play roles in root elongation. For example, a rhamnogalacturonan II xylosyltransferase (RG-II) gene, MGD4, participated in the growth of pollen tube and root by acting on pectic RG-II biosynthesis pathway [5]. In this study, qRT-PCR and GUS analyses also showed that AtEXPA4 was highly expressed in roots. Furthermore, it was confirmed that AtEXPA4 affected primary root elongation positively by comparing the primary root length of atexpa4 and AtEXPA4OE at different time points after seed germination. In addition, the growth rate of atexpa4 primary root sharply decreased from the 5th day to the 7th day after germination, suggest- ing that AtEXPA4 exerted the greatest effect during this period. We speculated that this effect might be related to the gradual increase of AtEXPA4 expression with the growth and development of primary roots. (Figure1D). However, the mutation and overexpression of AtEXPB5 did not affect the growth and development of the primary roots (Figure S9). This indicates that AtEXPA4 plays a major role in the growth and development of primary roots, and AtEXPB5 does not affect root growth because of its extremely low expression in roots. Generally, abnormal root system also affects the response of plants to abiotic stress. The overexpression of Triticum aestivum EXPB23 (TaEXPB23) showed increased lateral roots and higher root biomass, as well as enhanced drought tolerance [57]. Additionally, the analysis of AtEXPA4 and AtEXPB5 promoters showed that they both contained abundant response elements of abscisic acid, ethylene, and jasmonic acid (Figure S10), suggesting that they might participate in plant responses to abiotic stress. Further research is needed to confirm this.

5. Conclusions We isolated and characterized two expansin genes, AtEXPA4 and AtEXPB5, which are strongly expressed in mature pollens and pollen tubes. The molecular functions of AtEXPA4 and AtEXPB5 were analyzed by CRISPR/Cas9-mediated knockout and self- promoter-mediated overexpression. The results indicated that AtEXPA4 and AtEXPB5 are redundantly required for pollen tube growth. This enriches the role of expansins in the reproductive development of plants, and also shows that the genes of EXPA and EXPB subfamily can coordinate with each other to regulate plant growth and development. Furthermore, AtEXPA4 also showed a higher expression level in roots. Based on the statistics of primary root length and root meristem size, we found that AtEXPA4 has Genes 2021, 12, 249 13 of 16

a positive effect on the growth of primary roots. This also provides evidence for the involvement of expansin in the growth and development of plant roots. In addition, since there are many MYB binding sites and a variety of phytohormone response elements in the promoters of AtEXPA4 and AtEXPB5, future research can start from these aspects to uncover the regulatory pathways of expansin genes on plant growth and development.

Supplementary Materials: The following are available online at https://www.mdpi.com/2073-4 425/12/2/249/s1, Figure S1: Amino acid sequences of AtEXPA4 and AtEXPB5. The blue, red, and green letters represent signal peptide, DPBB_1 domain, and Pollen_allerg_1 domain, respectively. Figure S2: Subcellular localizations of AtEXPA4::eGFP and AtEXPB5::eGFP fusion proteins in tobacco leaf epidermal cells. Figure S3: Structures of CRISPR/Cas9 (A) and AtEXPA4 (B) AtEXPB5 (C) overexpression binary vectors for Arabidopsis thaliana transformation by floral dip method. The hSpCas9 cassette is driven by YAO promoter, while sgRNAs are controlled by AtU6-26 promoters. NLS, nuclear localization sequence. Figure S4: Sequence alignment of atexpa4 and atexpb5 mutants showed evidence of successful gene editing in the target regions, respectively. Figure S5: Sequence alignment of atexpa4expb5 mutants showed that the lines with both AtEXPA4 and AtEXPB5 knockout sites were successfully screened. (A), Detection of editing sites of AtEXPA4.(B) Detection of editing sites of AtEXPB5. Figure S6: Morphological observation of transgenic plants and floral organs. The first row is the overall shape of transgenic plants, scale bars, 4 cm, and the upper right corners are corresponding siliques, scale bars, 4 mm. The second row is the overall shape of flowers. The third row is the shape of pistils and stamens, scale bars, 1mm. Figure S7: Pollen morphology in atexpa4, atexpb5, atexpa4expb5, AtEXPA4OE, and AtEXPB5OE. Cytological staining observation includes Alexander staining, scale bars, 100 µm, DAPI staining, scale bars, 50 µm, and aniline blue staining, scale bars, 50 µm. SEM is used to observe pollen grain morphology and surface decoration, scale bars, 40 µm. Values are means, error bars are SD, n = 12 pistils per replicate, three biological replicates. Figure S8: Overexpression of AtEXPA4 and AtEXPB5 did not affect pollen elongation. (A) Aniline blue staining of pollen tubes at 3 h, 12 h, and 24 h after pollination in AtEXPA4OE lines and AtEXPB5OE lines, hours (h). Asterisks show positions where pollen tubes arrive, scale bars, 300 µm. (B) the percentage of pollen tube to pistil length in (A). Values are means, error bars are SD, n = 12 pistils per replicate, three biological replicates. Figure S9: AtEXPB5 does not affect the primary root elongation. (A) AtEXPB5OE and atexpb5 seedlings germinated and grown for 3, 5, and 7 days, scale bars, 2 mm. (B) Root lengths in (A). (C) Root growth rates from the 3rd day to the 5th day and from the 5th day to the 7th day after seed germination. Values are means, error bars are SD, n ≥ 12 seedlings per replicate, three biological replicates. Figure S10: Distribution of cis-acting elements in the 1.5 kb upstream promoter regions of AtEXPA4 and AtEXPB5. The different types of cis-acting elements are represented by different colors. The scale above was to measure nucleotides length. The results are predicted by the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Table S1. Primers used in this study. Table S2. Off-target detection of atexpa4. Table S3. Off-target detection of atexpb5. Author Contributions: Conceptualization, W.L. and J.C.; methodology, W.L.; validation, W.L. and L.X.; formal analysis, W.L. and L.X.; investigation, W.L. and H.L.; resources, J.C.; data curation, W.L.; writing—original draft preparation, W.L.; writing—review and editing, L.X. and J.C.; visualization, W.L. and H.L.; supervision, J.C.; project administration, W.L. and J.C.; funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grant from the National Natural Science Foundation of China (No. 31772311). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing not applicable. Acknowledgments: Thanks to Nianhang Rong from the Bio-ultrastructure Analysis Laboratory of Analysis center of Agrobiology and Environmental Sciences in Zhejiang University for supporting the use of scanning electron microscopy in this study. Conflicts of Interest: The authors declare no conflict of interest. Genes 2021, 12, 249 14 of 16

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